A variety of compounds may affect the rate at which existing biologic functions proceed. They are developed to speed up or slow down biological processes, e.g. by modulating enzyme activity or the binding of interacting molecules.
A compound's action is affected by the degree of attraction to its target (target affinity) and, once bound to the target, its ability to induce an effect (intrinsic activity or efficacy). Various compounds show various target affinity and efficacy.
Important classes of compounds functioning as modulator molecules are nuclear receptor interacting ligands.
The nuclear hormone receptor (further referred to as “nuclear receptor” or “NR”) super family comprises approximately 50 members that play a role in a broad range of physiologic processes, including metabolism, homeostasis, and reproduction. The classical NR proteins contain a central DNA-binding domain (DBD), which binds the promoter region of target genes, and a COOH-terminal ligand-binding domain (LBD). One of the functions of the LBD is co-regulator recruitment. Ligand binding induces a conformational change of the LBD. Subsequent binding to a co-activator or co-repressor protein then results in NR transactivation or the switch between a transcriptionally inactive state and an activated state of the receptor. Depending on the nature of the co-regulator, being either a co-activator or -repressor, an initiation point for the gene transcription machinery is respectively formed or blocked, and the target gene is activated or silenced. In addition, activated nuclear receptors can also alter pathways in the cytoplasm, such as kinase phosphorylation cascades.
As NRs respond to small ligand molecules or compounds and correspond to potent regulators of cell function, life and death, they are particularly attractive targets for the design of novel therapeutic agents. Small molecular ligands include biogenic amines, amino acids, ions, lipids, nucleotides, and chemical compounds that represent the majority of classic drugs.
A critical step in the design process of a therapeutic compound is the elucidation of its function. In the context of nuclear receptor interactions this function can be deduced from the modulation of receptor affinity for a co-activator or co-repressor and the resulting effect such as the transcriptional activity of the NR.
Methods to measure compound effects on nuclear receptor affinity for a co-regulator are known in the art. For example, Zhou et al (Mol. Endo. 1998, Vol. 12 (10), p. 1594-1604) describes the characterization of NR affinities for co-activator-derived domains by fluorescence resonance energy transfer (FRET). With this method, the ligand potency, calculated as EC50, is determined by quantification of the binding at equilibrium between nuclear receptor and co-activator at multiple ligand concentrations. However the multiple data points needed (for each variation in NR, co-regulator, ligand and their respective concentration, a separate sample and measurement is needed) makes this method by Zhou et al rather cumbersome and time and sample consuming.
Alternative methods in the art are described e.g. by Iannone et al (Cytometry 2001, Vol. (44), p 326-337) relating to the characterization of ligand function by measuring NR-LBD binding to a set of fluorescent microsphere-immobilised peptides with an LxxLL motif (in which L represents leucine and x can be any amino acid) the minimal entity in co-regulator proteins for interaction with a NR. Interaction is measured at a saturating ligand concentration while receptor concentrations are varied in separate samples. From these data an apparent Kd (affinity) of the receptor for each peptide is calculated. Other known motifs include but are not limited to LxxML, FxxFF or LxxIL (in which F represents phenylalanine, M represents Methionine, I represents isoleucine and x can be any amino acid).
Iannone et al (Mol. Endo. 2004, Vol. (18), p 1064-1081) also described the clustering of a set of ligands based on their profile of enhancement and or decrease of NR binding to individual peptides using saturating ligand concentrations by applying the same method as described above.
Again by applying the same method as described above Iannone et al (Cytometry Part A, 2006, Vol. (69A), p 374-383) also assessed the apparent affinity (Kd) by varying the density of the immobilized LxxLL peptides using a single NR concentration.
Disadvantages of the methods known in the art is that in most assays, each variation in NR, co-regulator, ligand and their respective concentration to either determine compound potency or efficacy requires measurements in separate samples and separate assays. The ability to perform such studies are limited by time, costs, reproducibility and availability of the bio-molecules such as the NR proteins, which are often difficult to obtain in high purity at large quantities.
Further, compound efficacies are calculated from NR-co-regulator binding in absence and presence of ligand (at a saturating concentration). As such the dynamic range of the readout is limited by the fact the maximal binding is measured when either one of the free binding partners (NR or co-regulator) is depleted. This limits the discrimination of high-efficacy binding-enhancing ligands.
The present invention overcomes the above-mentioned disadvantages by providing a multiplex method allowing the determination of both a compound's potency and efficacy in a time and cost effective way. In particular, the present invention provides a multiplex method for the determination of the potency and efficacy of nuclear receptor ligands in function of a kinetic reading of receptor-co-regulator binding.